(1) To develop animal disease (end host) and persistence (reservoir host) models: Over the past years we have developed and characterized rodent and nonhuman primate disease models for infections with influenza A viruses, the Middle East Respiratory Syndrome Coronavirus (MERS-CoV), henipaviruses (Nipah and Hendra), and hantaviruses causing Hantavirus Pulmonary Syndrome (HPS). The two nonhuman primate MERS models, rhesus macaque and common marmoset, were further refined with a comparative pathology study. The results suggested that increased virus replication and the local immune response to MERS-CoV infection play a role in the severity of pulmonary pathology. We also investigated whether domestic pigs could be used as an alternative MERS model. Pigs were inoculated intranasally and intratracheally with a high dose of MERS-CoV but did not develop signs of disease nor lesions in the respiratory tract. (studies ongoing) We have also further characterized the rhesus macaque HPS model to investigate mechanisms of disease pathogenesis. We are currently studying specific inflammatory events to gain understanding of what contributes to disease as well as to develop a therapeutic. Much of our work on hantaviruses in the past year was aimed at determining how the natural reservoirs are able to support high levels of virus replication without disease. We have previously shown that Sin Nombre virus elicits an initial inflammatory response in deer mice, the natural reservoir, but this response turned into an active anti-inflammatory response, as indicated by the activity of virus-specific T regulatory cells. Currently we are determining the requirement of these T regulatory cells to the suppression of the anti-inflammatory response by using T cell depletion strategies. (studies ongoing) We could demonstrate that Laguna negra infection causes HPS in Turkish hamsters. (finished and published) (2) To identify and characterize determinants of viral pathogenicity to develop antivirals: Severe influenza virus infections are often associated with bacterial co-infections. In order to study a potentiating effect of co-infection we performed a study in cynomolgus macaques using a moderately severe pandemic H1N1 strain (Ca04) and Methicillin-resistant Staphylococcus aureus (MRSA). Animals infected with MRSA only were largely asymptomatic, whereas animals infected with Ca04 only developed moderate pulmonary disease. Interestingly, animals initially infected with MRSA followed by Ca04 showed a dramatic reduction in clinical signs, whereas those initially infected with Ca04 showed enhanced clinical disease. Similar studies were performed with a seasonal H3N2 virus and MRSA, in which we did not see disease reduction or enhancement. Studies to decipher the mechanisms behind these observations were objectives over the past year and are still ongoing. (studies ongoing) Comorbidities are frequently reported in severe MERS cases. Thus, we tested the effect of immunosuppression on outcome of MERS-CoV infection in the rhesus macaque model. Immunosuppressed macaques did not develop more severe disease than immunocompetent animals, but they shed more virus, and viral loads in the lungs were significantly higher. Despite the increased virus replication, and in line with lack of increase in clinical disease, histological examination of the lungs showed a reduced inflammatory response in immunosuppressed macaques as compared to normal animals. These results suggested that the immune response to infection plays an important role in MERS-CoV pathogenesis. (studies ongoing) A novel henipavirus, Cedar paromyxovirus (CedPV) has not been associated with human disease. To elucidate mechanisms of disease induced by Nipah virus, we have used the hamster model of henipavirus disease to characterize and compare infection and pathogenesis. CedPV replicated in hamsters, yet did not cause any conspicuous pathology. In vitro studies suggest that CedPV elicits a strong innate immune response, whereas Nipah antagonizes this response, allowing for unchecked virus replication, resulting in massive cytopathology. These studies will continue and hopeful provide new insight into henipavirus pathogenesis that can be used to develop antivirals/therapeutics. (studies ongoing) (3) To identify and characterize host responses to viral infection to develop therapeutics: In collaboration with the Molecular Targets Program at NCI, griffithsin, a novel viral entry inhibitor, was identified as having potent (EC50 5nM) activity against MERS-CoV. The post-exposure efficacy of nebulized griffithsin in the rhesus macaque model showed moderate reduction of viral load but did not significantly reduce disease signs. We are currently refining the treatment scheme. We have also tested efficacy of three monoclonal antibodies (mAb) as a treatment for MERS-CoV infection in the common marmoset. These mAb had shown efficacy in mouse models of MERS-CoV infection. Unfortunately, none of the mABs showed significant reduction in disease burden and viral lung load in the nonhuman primate model suggesting that treatment with mABs may likely not very efficacious. Confirmatory studies and treatment with mAB cocktails are either ongoing or planned. Finally, we have tested the efficacy of the antiviral compound GS-5734 against MERS-CoV in the rhesus macaque model. Preliminary data indicate potent efficacy with reduction in disease burden and viral lung load. GS-5734 (Gilead) might be a promising treatment strategy for MERS. Confirmatory studies in the common marmoset model are planned. (4) To develop protective vaccines: We continued with our efforts to develop a universal vaccine against influenza A viruses. We currently are applying two approaches: i) expression of highly conserved B cell epitopes from two separate helical regions within the hemagglutinin stalk that have shown to afford heterosubtypic binding and protection, and ii) removal of hemagglutinin globular region to increase antibody responses against otherwise poorly antigenic epitopes. We used the Cytomegalovirus (CMV) vector platform for these studies, which is able to induce long-lasting immune responses (both T cell and antibody). Unfortunately, first attempts using the mouse model of influenza A viruses were rather discouraging. We will continue to optimize the CMV platform but have also started to use the vesicular stomatitis virus (VSV) as an alternative platform. (studies are ongoing) For MERS, we have obtained very promising results with a DNA vaccine platform encoding a codon-optimized consensus spike protein. This vaccine candidate induced potent cellular immunity and antigen specific neutralizing antibodies in three animal species, mice, macaques and camels using a prime/boost/boost approach. Vaccinated macaques were protected against MERS-CoV challenge and did not show any clinical or radiographic signs of pneumonia. Recently, we were successful in shortening the vaccination strategy (prime/boost) for potential application of this vaccination approach in emergency situations to prevent MERS-CoV infection. (published and still ongoing) In an effort to generate an effective countermeasure for Nipah virus infection, we used the VSV based vaccine approach to express one of the glycoproteins of Nipah virus as an immunogen. These vaccines elicited strong antibody responses in hamsters and nonhuman primates and protected them from lethal, high dose Nipah virus challenge. We could demonstrate that not only is a strong neutralizing response elicited, but the vaccine primes the CD8+ T cell response as well. To investigate the limits of the efficacy of this vaccine, we used the hamster model and showed that this vaccine provides at least partial protection when given as late as on the day of virus challenge (DeBuysscher et al., NPJ Vaccines: accepte